The development of embryos reconstructed by somatic cell nuclear transfer (SCNT) is dependent upon numerous factors. Central to development is the quality and developmental competence of the recipient cytoplast and the type of the donor nucleus. Typically metaphase of the second meiotic division (MII) has become the cytoplast of choice. Production of a cytoplast requires removal of the recipient genetic material, however, it may remove proteins which are essential for development or reduce the levels of cytoplasmic proteins to influence subsequent reprogramming of the donor nucleus. In this study, enucleation at MII did not affect the activities of either MPF or MAPK kinases. Immunocytochemical staining showed that both Cyclin B1 (MPF) and Erk1/2 (MAPK) were associated with the meiotic spindle of AI/TI oocytes with little staining in the cytoplasm, however, at MII association of both proteins with the spindle had reduced and a greater degree of cytoplasmic distribution was observed. The analysis of oocyte proteins removed during enucleation is a difficult approach to the identification of factors which may be depleted in the cytoplast. This is primarily due to the large numbers of aspirated karyoplasts which would be required for the analysis.

ABSTRACT   INTRODUCTION   MATERIALS AND METHODS    Collection and Culture of Sheep Oocytes    Assessment of MPF and MAPK Activities    Immunocytochemistry    Enucleation    Electrophoretic Separation of Proteins    Statistical Analysis   RESULTS    MPF and MAPK Activities at Different Stages of Maturation    Localization of Cyclin B1 and Erk1/2 in Ovine Oocytes    MPF and MAPK Activities of Cytoplasts and KaryoplastsFollowing Enucleation    Determination of Proteins Depleted from Oocytes Following Enucleation   DISCUSSION   REFERENCES

• Joon-Hee Lee(Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University, Division of Animal Physiology, School of Biosciences, The University of Nottingham, Sutton Bonington,Institute of Agriculture & Life Science, College of Agriculture and Life Sciences, Gyeongsang National University) Corresponding author
• Keith H. S. Campbell(Division of Animal Physiology, School of Biosciences, The University of Nottingham)